Naked Science Forum

(Not sure if this is the right forum but unsure where else to post) Hi, I'm in a team investigating CRISPR/Cas9 editing in wheat. We were wondering why we didn’t have any edits in our wheat plants using CRISPR/Cas9 technology and what we could possibly do to ensure we would have edits in the future?

So, our experiment consisted of:

40 plants that had been successfully transformed using biolisitcs with a Cas9 construct and sgRNAOf these we genotyped to find out which had Cas9 DNA, and of these which were expressing Cas9 mRNA, we used PCR to determine thisWe then worked out which of the 40 plants had the sgRNAThe plants that had both sgRNA and Cas9 we took forward and sequenced using Next Generation Sequencing to determine if the CRISPR/Cas9 construct had performed any edits (insertions or deletions)

We found no editing in any of the plants, but unsure why this would be the case and what we could do to ensure editing in the future?

Ideas I’ve identified so far:

The Cas9 mRNA wasn’t translated properly: we’d test this doing a western blot?The wheat genome is heavy in repeat regions and has three genomes (hexaploid) so the DNA was repaired more easily therefore any edits were simply repairedWhen checking for Cas9 fragment not the entire Cas9 gene had been inserted: in future we’d test for entire gene using long range PCR

Any help or ideas would be hugely appreciated, a bit frustrating we didn't find any edits so now trying to work out why, and what we could do to ensure we'd get some in the future.

2) Maybe the strand-breaks introduced by CRISPR were repaired using a complementary sequence from the other parallel genome, as you surmise, rather than the guide sequence?

- there is a precedent for a strange thing like 2) happening:

When scientists "fixed" human embryos carrying a defect in a gene linked to a form of developmental heart condition, they found that the error was fixed from the complementary region of the corresponding healthy chromosome, rather than the supplied patch. Might this happening in your plants?https://www.thenakedscientists.com/articles/science-news/gene-editing-corrects-embryonic-heart-defect

Thanks for sending that article as well that is very interesting and I hadn't come across that at all, the only worrying thing is that our CRISPR/Cas9 constructs should only have edited using NHEJ which is itself a 'repairing' mechanism, all be it a faulty one, not HDR. Or was that not what you mean?

2) Maybe the strand-breaks introduced by CRISPR were repaired using a complementary sequence from the other parallel genome, as you surmise, rather than the guide sequence?

- there is a precedent for a strange thing like 2) happening:

When scientists "fixed" human embryos carrying a defect in a gene linked to a form of developmental heart condition, they found that the error was fixed from the complementary region of the corresponding healthy chromosome, rather than the supplied patch. Might this happening in your plants?

Thanks for sending that article as well that is very interesting and I hadn't come across that at all, the only worrying thing is that our CRISPR/Cas9 constructs should only have edited using NHEJ which is itself a 'repairing' mechanism, all be it a faulty one, not HDR. Or was that not what you mean?

2) Maybe the strand-breaks introduced by CRISPR were repaired using a complementary sequence from the other parallel genome, as you surmise, rather than the guide sequence?

- there is a precedent for a strange thing like 2) happening:

When scientists "fixed" human embryos carrying a defect in a gene linked to a form of developmental heart condition, they found that the error was fixed from the complementary region of the corresponding healthy chromosome, rather than the supplied patch. Might this happening in your plants?